Hybrid Nanochannel Arrays for Single Molecule Linear Genome Analysis Abstract The objective of this proposal is to develop a fully integrated nanofluidic device for single molecule analysis of long, linearized, native state genomic DNA. The project will result in a simple, disposable chip that will allow direct, fluorescent single molecule analysis of structural, CNV, haplotype and epigenomic variations at the single molecule level. The resulting commercial system will permit researchers and clinicians to directly visualize genomic variations from a few kilobases to megabases in length, from as little as a single cell to common bulk preparation methods, on common microscope imaging systems. Advances in technologies to analyze complex genomes have allowed for significant collection and analysis of a wide range of genetic data including gross structural variation, repetitive elements, genotypes, haplotypes and more generically, the DNA sequence. But current technologies suffer from relatively low sensitivity and specificity due to data generated from averaged heterogeneous bulk sample preparations or difficulties in standardizing and scaling up the analytical platform and biochemical protocols. The proposed hybrid micro/nanofluidic device will allow isolation, manipulation and direct imaging of single molecule features without significantly changing upstream biochemical process of the samples. Leveraging high quality mass micro/nanofabrication technology developed by the semiconductor industry, these devices could be made truly affordable to end users while allowing consistent and comparable results on a standardized format. Based upon phase I results, we will optimize a nearly universal, high resolution single molecule level DNA analysis device. The specific aims of phase II are to 1) develop fluorescent standards and sample buffer for evaluating device performance, 2)develop a simple, disposable chip cartridge that can be easily loaded by standard pipetting, 3) develop a modular joystick control system for manipulating the flow of DNA within the device while mounted on standard microscopy systems. Completion of this project will result in an integrated biochip device capable of providing statistically significant, single-molecule-level analysis of megabase DNA with sub-kilobase resolution. This technology could greatly enhance our ability to understand many different types of genetic variations and of genomic instabilities related to cancer and other genetic diseases with consistent and reproducible performance that will enable broad incorporation of single molecule analysis into diagnostic and medical genetics applications. PUBLIC HEALTH RELEVANCE: The development of an open architecture, single molecule, linear DNA analysis system will provide researchers and clinicians alike a new tool for genomic investigation, providing new insights into the complexity of human genetic disease and genome structure. The availability of an inexpensive, disposable nanodevice compatible with common microscopy systems will enable broad access to single molecule research of human genome variation, such as structural mapping, haplotyping and even copy number variation, as well as the characterization of overall genomic state. This will help expand the use of genomic analysis as a tool for understanding complex disorders and will help enable the realization of practical, cost effective personalized medicine.